Functional Characterisation of Two Channels Proteins Involved in Leguminous Symbiosis

Legume-rhizobial symbiosis results in the formation of a new organ,
the nitrogen-fixing root nodule. A chemical communication between
both partners accompanies the invasion of plant host cells by
bacteria and the development of the root nodule. In response to
plant-released flavonoids, rhizobia produce
lipo-chito-oligosaccharide signalling molecules, so called Nod
factors. Early signal transduction in legumes such as Lotus
japonicus and Medicago truncatula, is associated with a succession
of tightly orchestrated ion fluxes across different membrane
systems of the host cell. The Nod factor perception at the plasma
membrane triggers Ca2+ oscillations that are associated with the
nucleus. CASTOR and POLLUX are required for Ca2+ spiking. Homology
modeling suggested CASTOR and POLLUX might be ion channels.
However, experimental confirmation was lacking. Therefore we
performed biochemical and electrophysiological analysis to define
their role. Here we show that CASTOR and POLLUX form two
independent homocomplexes in the nuclear rim in planta. We
reconstituted CASTOR in planar lipid bilayers and
electrophysiological measurements revealed that CASTOR is a cation
channel preferentially permeable to potassium. The permeability of
the sequence-related POLLUX for cation could be as well
demonstrated through expression of POLLUX in different yeast
mutants. Furthermore, we demonstrate that a voltage-dependent
magnesium blocking mechanism contributes to reduce the conductance
of CASTOR at negative membrane potential. By screening a L.
japonicus roots cDNA library using yeast-two-hybrid system, a SNF7
protein interacting with CASTOR was found which acts as positive
regulator in the nodulation pathway. Collectively the data
demonstrate that both CASTOR and POLLUX are nuclear localized
cation channels. Therefore, we propose that CASTOR and POLLUX may
act as counter ion channels to facilitate a rapid efflux of charge
associated with the calcium efflux. Alternatively and not mutually
exclusive, they may catalyze a nuclear membrane depolarization
leading to the activation of calcium channels responsible for
calcium spiking